Eukaryotic proteins having clinical or industrial value may be obtained in large quantities using techniques which facilitate their synthesis in bacteria or in eukaryotic cell cultures. However, once synthesized, there are often problems recovering these recombinant proteins in substantial yields and in a useful form. Recombinant proteins expressed in bacteria often accumulate in the bacterial cytoplasm as insoluble aggregates known as inclusion bodies (F. A. O. Marston, Biochem. J. 240:1-12 (1986); C. H. Schein, Biotechnology 7:1141-1149 (1989)). In order to effectively utilize the protein for immunochemical manipulations, the protein must be recovered in a soluble form which is immunologically active.
Proteins have been solubilized using guanidinium salts, urea, detergents, or other organic solvents. However, the efficacy of the solubilizing agent appears to vary along with the physical characteristics of the protein. It also has been suggested that 0.01-2% Triton or SDS detergents can also be used to denature inclusion body proteins, (Bruggeman et al., Biotechniques 10:202-209 (1991)), however, this concentration range is insufficient for very insoluble inclusion body proteins. There is no general method of solubilization that works for most proteins. (D. L. Wilkinson and R. G. Harrison, Biotechnology 9:443-448 (1991); F. A. O. Marston, in: DNA Cloning: A Practical Approach, Vol. III, (D. Glover, ed.), IRC Press pp. 59-88 (1989)).
Some techniques for solubilizing inclusion body proteins initially require a strong denaturing solution to solubilize the protein and subsequently require at least a non-ionic, weaker denaturing solution to maintain the protein solubility. (Builder & Ogez, U.S. Pat. No. 4,511,502 (1985); Olson, U.S. Pat. No. 4,518,526 (1985); Olson & Pai, U.S. Pat. No. 4,511,503 (1985); Jones et al., U.S. Pat. No. 4,512,922 (1985)).
The use of guanidinium salts and urea for solubilizing proteins has two problems. The first is that unless the protein concentration is dilute, the protein precipitates out of solution when the solubilizing agent is removed (J. Krueger et al., BioPharm. 2:40-45 (1989)). Solutions having protein concentrations of more than one milligram per milliliter are generally not handled successfully by this method (F. A. O. Marston, in: DNA Cloning: A Practical Approach, Vol. III, (D. Glover, ed.), IRC Press pp. 59-88 (1989)). The second problem is that use of guanidinium salts and urea precludes subsequent chemistry involving binding of a protein's reactive amine groups, since the urea and guanidinium salts have reactive primary amines that swamp those of the protein. One chemical method that requires reactive amine groups is the preparation of affinity matrices that bind a protein to an inert support by a chemical bond between a primary amine group on the protein and an activated group on the support.
Recombinant transmembrane proteins which contain both hydrophobic and hydrophilic regions are especially intractable to solubilization. The protein gp41 is a component of the viral envelope of the human immunodeficiency virus (HIV) and is useful for the sensitive and specific detection of anti-HIV antibodies. Recombinant gp41 from E. coli inclusion bodies is extremely insoluble. The recombinant protein is insoluble at pH 8.9 in 8M urea or 7M guanidine-HCl, as well as in the non-ionic detergents 1.5% octyl B-D-glucopyranoside or 2% Triton X-100. To substantially solubilize the protein requires 8M urea or 6M guanidinium hydrochloride at pH 12.5. (Soutschek et al., J. of Chrom. 521:267 (1990)). This method would not meet the standard of safety required for injection of materials into humans because urea or guanidinium salts are required to maintain protein solubility but are not suitable for human injection. Therefore, this procedure would not be appropriate for recovering recombinant protein antigens for human vaccines against this AIDS virus component.
Additionally, biological products intended for injection must be free of bacterial endotoxins, the lipopolysaccharide component of the cell walls of gram negative bacteria. Endotoxin can produce pyrogenic and shock reactions after systemic administration. Therefore, where inclusion body protein is intended for systemic administration in humans or animals, the protein preparation must be in a form that can be treated to remove endotoxins.
What is needed is a protein solubilization method which maintains the protein in solution after removal of the solubilizing agent and preserves the immunological activity of the protein. Additionally, a method is needed whereby protein solubility is stabilized without the need for amine-containing denaturants which interfere with protein reactivity. Still further, a method is needed which is efficient for proteins having widely varying solubilities and adequate for very insoluble proteins.